The blood and other body fluids contain free circulating DNA, also known as cell-free DNA (cfDNA). In cancer patient the amount of cfDNA in plasma and serum samples can be elevated a 1000-fold or more. The level of cfDNA can thus be used both as a diagnostic and prognostic marker for cancer as well as a marker for monitoring the response of the tumor to treatment (Fleischhacker and Schmidt, Cancer Biomark. 2010; 6(3-4):211-9; Goebela et al., Disease Markers 2005; 21:105-120). In addition, the cfDNA of an individual suffering from or at risk for cancer can be analyzed for the presence of known tumor-specific markers, aiding in the diagnosis, staging and prognosis of the disease (Spindler et al., Clin Cancer Res 2012; 18:1177-1185; European Patent 1 712 639 B1; Dawson et al., N Engl 3 Med. 2013; 368(13):1199-209). Analysis of cell-free fetal DNA present in maternal blood provides a method of non-invasive prenatal diagnosis of the fetus, and circulating free plasma DNA has been implicated in conditions associated with tissue injury, including exercise-induced inflammation, and thus is a potential marker for athletic overtraining (Fatouous et al., Clin Chem 2006; 52(9):1820-4).
Since the concentration of cfDNA may, however, be very low, including controls for pre-analytical pitfalls are of utmost importance. One particular problem is that the plasma and serum samples may become contaminated with cellular DNA from blood cells, typically lymphocytes, ruptured during or after the taking of the sample. For example, hemolysis may occur during drawing of the blood sample as a result of the pressure drop from vein to the collection tube, and the cells may lyse during prolonged storage times prior to isolating plasma or serum or because of profound pipetting when isolating the supernatant. Whatever the reason, the cfDNA is then contaminated with DNA from lysed lymphocytes, resulting in falsely increased levels of cfDNA. This increases the risk for, e.g., false positive results when comparing the level of cfDNA to a control value. Contamination may also introduce other errors, such as falsely low levels when monitoring for a cancer-specific mutation and normalizing its level to the total cfDNA in the sample.
U.S. 2010/0124743 A1 relates to a method of diagnosing cancer based on analysis of free DNA in plasma where the measured level of free DNA is compared to first and second threshold values, with the second threshold value being the highest one. If the free DNA is higher than the second threshold value, it is determined that the plasma free DNA is contaminated by normal cell-derived DNA from, e.g., lymphocytes.
WO 2006/128192 relates to the use of cfDNA for diagnosing cancer based on analysis of ALU and LINE repeats in serum. The problems of loss of DNA and contamination by DNA released from cells present in the blood are addressed by eliminating unnecessary purification steps.
US 2013/0012405 A1 relates to methods for diagnosing e.g. cancer by screening for biomarkers in circulating microRNAs. Finding that the measurements were frequently confounded by cellular miRNAs of different hematopoietic origin, cellular miRNA signatures were catalogued and analyzed.
Jahr et al. (2001) describes the quantification of the fraction of plasma DNA derived from tumor cells by quantitative methylation-specific PCR, and analyses the possible origins of non-tumor DNA in blood. They considered the possibility that degenerating tumor-infiltrating T lymphocytes contributed to the DNA levels in blood, and therefore quantified the presence of T-cell DNA in plasma samples by PCR amplification of a region the T-cell receptor beta-chain, which exhibits a somatic rearrangement by VDJ recombination.
Ivancic-Jelecki et al. (2009) describes a method for DNA isolation by using anion-exchange chromatography and its use in determining the concentration of contaminating DNA in plasma.
Despite these advances in the art, there is still a need for a control analysis enabling identification of serum and plasma samples comprising contaminating DNA from normal blood cells.